CD150­dependent activation of EBV­transformed B cells induces the differentiation of peripheral blood monocytes via the secretion of multiple cytokines.

CD150, also termed signaling lymphocyte activation molecule family member 1, is a cell surface receptor expressed on T cells, B cells, dendritic cells (DCs) and some tumors. Stimulation of CD150 on immune cells induces cell proliferation and cytokine production. However, the function of CD150 in Epstein­Barr virus (EBV)­infected B cells is still not fully understood. In the present study, CD150 expression on B cells increased rapidly following EBV infection, and various CD150 antibodies, measles viral proteins and recombinant CD150 proteins induced the secretion of multiple cytokines in both CD150+ EBV­transformed B cells and EBV+ lymphoma cells. Notably, the IL­1α protein level showed the greatest increase among all cytokines measured. The culture supernatant containing these cytokines induced the rapid differentiation of monocytes to DCs after only 2 days in vitro, which was faster than the established DC maturation time. Furthermore, knockdown of CD150 expression led to a reduction in the secretion of multiple cytokines, and monocyte differentiation was partially inhibited by anti­IL­1α and anti­granulocyte­macrophage colony­stimulating factor neutralizing antibodies. Collectively, the results of the present study suggest that CD150 activation triggers cytokine production in EBV­transformed B cells, and that measles virus coinfection might affect immune responses through the production of various cytokines in EBV+ lymphoma cells.

Role of Peroxiredoxin 1 Induced by Epstein-Barr Virus Infection in Nasopharyngeal Carcinoma.

BACKGROUND/AIM: Nasopharyngeal carcinoma (NPC), a common cancer in Southern China, is associated with Epstein-Barr Virus (EBV) infection. Although many therapies for NPC have been established, the definite role of EBV in NPC remains unclear. Therefore, this work focuses on LMP2A, a latent EBV gene, and investigates whether LMP2A is related to peroxiredoxin 1 (PRDX1) in EBV-positive NPC. MATERIALS AND METHODS: The mRNA and protein expression levels of LMP2A, PRDX1, and beta-catenin were compared in patient samples. To identify molecular mechanisms, EBV-negative NP69 and EBV-positive C666-1 NPC cell lines were used. After making an agar cell block for cell slides, the intensity of LMP2A expression was observed visually. To measure the level of reactive oxygen species, both fluorescence microscope and flow cytometry were used. To investigate the intracellular signaling molecular mechanisms with and without the LMP2A gene, reverse transcription polymerase chain reaction and western blotting were used. RESULTS: Both patient samples and cells of nasopharyngeal carcinoma infected with EBV had increased expression of LMP2A compared with controls, and high ROS levels were identified. Cell viability assay showed that LMP2A promoted cell growth by regulating gene expression. Furthermore, LMP2A induced the expression of PRDX1 and beta-catenin. LMP2A also increased the expression of both cyclin B1 and cyclin D1. CONCLUSION: In NPC cells, PRDX1 and beta-catenin were regulated through LMP2A expression, which reduced cell growth through cell cycle-related gene expression. This study suggests that LMP2A could be a target molecule for inhibiting cancer progression in NPC cells infected with EBV.

Targeting isoforms of RON kinase (MST1R) drives antitumor efficacy.

Recepteur d'origine nantais (RON, MST1R) is a single-span transmembrane receptor tyrosine kinase (RTK) aberrantly expressed in numerous cancers, including various solid tumors. How naturally occurring splicing isoforms of RON, especially those which are constitutively activated, affect tumorigenesis and therapeutic response, is largely unknown. Here, we identified that presence of activated RON could be a possible factor for the development of resistance against anti-EGFR (cetuximab) therapy in colorectal cancer patient tissues. Also, we elucidated the roles of three splicing variants of RON, RON Δ155, Δ160, and Δ165 as tumor drivers in cancer cell lines. Subsequently, we designed an inhibitor of RON, WM-S1-030, to suppress phosphorylation thereby inhibiting the activation of the three RON variants as well as the wild type. Specifically, WM-S1-030 treatment led to potent regression of tumor growth in solid tumors expressing the RON variants Δ155, Δ160, and Δ165. Two mechanisms for the RON oncogenic activity depending on KRAS genotype was evaluated in our study which include activation of EGFR and Src, in a trimeric complex, and stabilization of the beta-catenin. In terms of the immunotherapy, WM-S1-030 elicited notable antitumor immunity in anti-PD-1 resistant cell derived mouse model, likely via repression of M1/M2 polarization of macrophages. These findings suggest that WM-S1-030 could be developed as a new treatment option for cancer patients expressing these three RON variants.

Bone destruction in chronic otitis media is not mediated by the RANKL pathway or estrogen receptor-alpha.

BACKGROUND: Chronic otitis media with or without cholesteatoma progresses with various degrees of bone resorption and remodeling. Estrogen mediates osteoprotective effects through the receptor activator of NF-κB ligand (RANKL) pathway, which is mainly mediated by estrogen receptor-alpha (ER-α). OBJECTIVES: The present study investigated the expression patterns of receptor activator of NF-κB (RANK), osteoprotegerin (OPG), RANKL, and ER-α in pathological tissue from patients with chronic otitis media to determine the roles of those factors in osteolytic mechanisms underlying the pathogenesis of chronic otitis media. METHODS: Normal and pathological specimens from 18 patients with chronic otitis media were examined. RESULTS: There were no significant differences in RANK, OPG, RANKL, or ER-α mRNA expression between normal and pathological specimens of epithelial tissue. CONCLUSIONS: Our findings suggested that RANK, OPG, RANKL, and ER-α are not associated with the bone destruction in chronic otitis media; other cytokines may directly activate the osteoclasts in chronic otitis media.

APX­115A, a pan­NADPH oxidase inhibitor, reduces the degree and incidence rate of dry eye in the STZ­induced diabetic rat model.

Dye eye disease (DED) is a common ocular disorder in patients with diabetes. It has been reported that APX-115A, a pan-nicotinamide adenine dinucleotide phosphate (NAPDH) oxidase inhibitor, has an apoptosis-inducing effect on Epstein-Barr virus-infected retinal epithelial cells, but its effects in DED are poorly understood. Therefore, a rat model of diabetes was used in the present study to investigate whether APX-115A has an impact on DED in diabetic rats. A diabetic model was established in male Sprague Dawley rats via the intraperitoneal injection of streptozotocin. The eyeballs of the rats were treated with a solution containing APX-115A or a saline control. Tear secretion was measured with the phenol red thread tear test, and the morphology of the eyeball and lacrimal gland tissues was determined using hematoxylin and eosin staining. In addition, localization of NAPDH oxidase 2 (NOX2) in the eyeball and lacrimal gland tissues was detected by immunohistochemistry. The APX-115A treatment had no effect on body weight, blood glucose level or the size of the lacrimal glands. However, morphological changes, namely intracellular vacuoles and acinar atrophy, were observed in the lacrimal glands of the diabetic rats, and APX-115A treatment attenuated these changes. Immunohistochemistry revealed that NOX2 expression was decreased in the lacrimal glands of the diabetic rats, and APX-115A treatment did not attenuate the reduction in NOX2. The corneas of the diabetic rats treated with APX-115A exhibited no change in thickness but had lower NOX2 expression levels compared with those of the control diabetic rats. APX-115A also increased tear secretion and ameliorated the histological changes associated with diabetes. Furthermore, the NOX2 expression levels in the corneas of the diabetic rats treated with APX-115A were restored to the levels observed in normal rats. These findings suggest that APX-115A has potential as a therapeutic agent for DED.

The Role of V-Set Ig Domain-Containing 4 in Chronic Kidney Disease Models.

V-set Ig domain-containing 4 (VSIG4) regulates an inflammatory response and is involved in various diseases. However, the role of VSIG4 in kidney diseases is still unclear. Here, we investigated VSIG4 expression in unilateral ureteral obstruction (UUO), doxorubicin-induced kidney injury mouse, and doxorubicin-induced podocyte injury models. The levels of urinary VSIG4 protein significantly increased in the UUO mice compared with that in the control. The expression of VSIG4 mRNA and protein in the UUO mice was significantly upregulated compared with that in the control. In the doxorubicin-induced kidney injury model, the levels of urinary albumin and VSIG4 for 24 h were significantly higher than those in the control mice. Notably, a significant correlation was observed between urinary levels of VSIG4 and albumin (r = 0.912, p < 0.001). Intrarenal VSIG4 mRNA and protein expression were also significantly higher in the doxorubicin-induced mice than in the control. In cultured podocytes, VSIG4 mRNA and protein expressions were significantly higher in the doxorubicin-treated groups (1.0 and 3.0 µg/mL) than in the controls at 12 and 24 h. In conclusion, VSIG4 expression was upregulated in the UUO and doxorubicin-induced kidney injury models. VSIG4 may be involved in pathogenesis and disease progression in chronic kidney disease models.

15-Prostaglandin Dehydrogenase Inhibition Enhances Colon Cancer Metastasis by Up-regulation of Epithelial-to-Mesenchymal Transition Genes.

BACKGROUND/AIM: Most deaths from colon cancer are due to metastasis. Recently, PGE2 was found to influence colon cancer invasion and metastasis. 15-PGDH, an enzyme that metabolizes PGE2, is known as a tumor suppressor in colonic carcinogenesis. This study investigated the effect of 15-PGDH on colon cancer metastasis. MATERIALS AND METHODS: 15-PGDH expression by immunohistochemical staining, clinicopathologic features, and 5-year cancer-specific survival were investigated in colon cancer patients. Liver metastasis was examined by assaying 15-PGDH activity in an animal model. Changes in PGE2, proliferation, migration, and invasion of the colorectal cancer cell line HCT116, were examined using a 15-PGDH inhibitor (SW033291) or enhancer (CDDO-ME). The expression of genes involved in the epithelial-to-mesenchymal transition (EMT) was also studied. RESULTS: The absence of 15-PGDH expression significantly correlated with advanced-stage, lymph node metastasis, and decreased cancer-specific survival in colon cancer patients. Inhibition of 15-PGDH increased colon cancer liver metastasis in the animal model. The 15-PGDH inhibitor, SW033291, increased PGE2 and decreased 15-PGDH expression on HCT116. However, treatment with CDDO-ME, a substance that enhances 15-PGDH, showed the opposite results. Inhibition of 15-PGDH increased cell proliferation, migration, and invasion, but activation of 15-PGDH showed the opposite effect. Inhibition of 15-PGDH also affected the EMT markers, N-cadherin, Snail, and Twist2. CONCLUSION: 15-PGDH inhibition increased colon cancer metastasis by inducing changes in EMT-related genes via an increase in PGE2 expression and could be a promising biomarker for anticancer treatment.

Pan-Aurora Kinase Inhibitor Danusertib Induces Apoptosis of Epstein-Barr Virus-transformed B-Cells Through Caspase and Endoplasmic Reticulum Stress Signaling.

BACKGROUND/AIM: Evidence for the relevance of Epstein-Barr virus (EBV) in various types of cancer has expanded; however, the definitive mechanism of EBV-induced oncogenesis remains ambiguous. The purpose of this study was to identify the relevance of aurora kinases in EBV-induced carcinogenesis, and the cellular responses to danusertib, a pan-aurora kinase inhibitor. The underlying signaling mechanism in EBV-transformed B-cells was also investigated. MATERIALS AND METHODS: Western blotting was performed on EBV-transformed B-cells and EBV-positive lymphoma cells to identify aurora kinase expression. Cellular responses of EBV-transformed B-cells to danusertib were investigated using AlamaBlue assay and apoptosis analysis. To evaluate the underlying signaling mechanisms of danusertib-induced apoptosis, cleavage of caspase cascade molecules, endoplasmic reticulum (ER) stress-associated molecule activation, and intracellular Ca2+ levels were evaluated using western blotting, flow cytometry, and inhibition assays. RESULTS: Expression of both aurora kinase A and B was gradually increased in EBV-infected B-cells and two EBV-positive B lymphoma cell lines. Danusertib significantly suppressed EBV-transformed B-cell proliferation in a dose-dependent manner. Danusertib induced apoptosis and cell cycle arrest through disruption of mitochondrial membrane potential in EBV-transformed B-cells in a dose-dependent and time-dependent manner. Moreover, danusertib induced cleavage of caspases, ER stress-associated molecule activation, and intracellular Ca2+ release from ER to cytoplasm in EBV-transformed B-cells, while BAPTA-AM, a calcium chelator, inhibited danusertib-induced apoptosis. CONCLUSION: Danusertib treatment led to apoptosis of EBV-transformed B-cells through ER stress-associated proteins and mitochondrial caspase activation. These results suggest that aurora kinases may be valuable targets for potential therapeutic agents against EBV-associated carcinoma.

Upregulation of VSIG4 in Type 2 Diabetic Kidney Disease.

Fibrosis is the final common finding in patients with advanced diabetic kidney disease. V-set Ig domain containing 4 (VSIG4) is related to fibrosis in several diseases. It also contributes to fibrosis under high-glucose conditions in renal tubule cells. To determine the role of VSIG4 in type 2 diabetes, we examined VSIG4 expression in a type 2 diabetic animal model and podocyte. Urinary excretion of albumin and VSIG4 was significantly higher in db/db mice than in the control group. Urine VSIGs levels for 6 h were about three-fold higher in db/db mice than in db/m mice at 20 weeks of age: 55.2 ± 37.8 vs. 153.1 ± 74.3 ng, p = 0.04. Furthermore, urinary VSIG4 levels were significantly correlated with urinary albumin levels (r = 0.77, p < 0.01). Intrarenal VSIG4 mRNA expression was significantly higher in db/db mice than in control mice (1.00 ± 0.35 vs. 1.69 ± 0.77, p = 0.04). Further, VSIG4 expression was almost twice as high in db/db mice at 20 weeks of age. Intrarenal VSIG immunoreactivity in db/db mice was also significantly higher than that in control mice. In cultured podocytes, both high glucose and angiotensin II significantly upregulated the expression of VSIG4 mRNA and protein. In conclusion, VSIG4 was upregulated in an animal model of type 2 diabetes and was related to albuminuria and pro-fibrotic markers. Considering these relationships, VSIG4 may be an important mediator of diabetic nephropathy progression.

A Case of Pathologically Confirmed Corticobasal Degeneration Initially Presenting as Progressive Supranuclear Palsy Syndrome.

Progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) overlap clinically with parkinsonism or extrapyramidal signs and pathologically with tauopathy. Asymmetric parkinsonism and cortical dysfunctions are classical features of CBD. However, symmetric parkinsonism, frequent falls, and supranuclear gaze palsy are key features of PSP. Despite biochemically classified as 4R tauopathies, tufted astrocytes of PSP and astrocytic plaque of CBD show pathologically important differences. Herein, we report a 68-year-old man with pathologically confirmed CBD. He was clinically suspected to have PSP because of progressive gait disturbances, frequent falls, and vertical saccade limitation. Neurological examination performed at age 71 revealed symmetrical bradykinesia, axial rigidity, and postural instability with worsening of early existing symptoms. Magnetic resonance imaging of the brain taken at age 70 detected midbrain and left frontotemporal atrophy and right middle cerebral artery infarction. Left frontotemporoparietal hypometabolism and asymmetrically decreased fluoro-propyl-carbomethoxy-iodophenyl-tropane uptake in the basal ganglia were observed. The autopsy was performed at the time of his death (at age 72), which revealed severe pallor of the substantia nigra and mildly hypopigmented locus ceruleus. AT8 immunohistochemistry and Gallyas staining revealed tau-positive neuronal and glial inclusions, astrocytic plaques, ballooned neurons, and numerous threads in both gray and white matter. No abnormal inclusions were revealed by beta-amyloid, α-synuclein and TDP-43 immunohistochemistry. In our case, cerebral infarction, periventricular and deep white matter ischemic changes, and midbrain atrophy were likely to produce PSP-CBD overlapping symptoms. However, our patient was finally confirmed to have CBD based on pathological findings such as astrocytic plaques.

One Autopsy Proved Neocortical Lewy Body Disease Without the Involvement of the Olfactory Bulb and Brainstem.

Lewy bodies (LBs) and Lewy neurites (LNs) are pathological hallmarks of Parkinson's disease (PD) or dementia with LBs (DLB). Incidental Lewy body disease (iLBD) is defined when LBs and LNs are found in the brain of normal elderly individuals. A 65-year-old man presented with autopsy-proven Lewy body pathology (LBP). He had never complained of cognitive impairments or parkinsonian motor symptoms, and he had always maintained independence in activities of daily living. Hypopigmentations in the locus coeruleus and substantia nigra were discovered during the autopsy. The patient showed severe-to-extremely severe LBs in the neocortex and limbic areas, except in the nucleus basalis of Meynert, amygdala, and brainstem, according to microscopic findings. Hence, using several of the previously known staging systems, it was difficult to classify the patient's LBP type. Furthermore, these findings were unique because they had never been observed before in iLBD.

Morphologic Changes in Esophageal Body Movement During Bolus Transport After Peroral Endoscopic Myotomy in Type III Achalasia.

BACKGROUND/AIMS: The effect of peroral endoscopic myotomy (POEM) on esophageal body movement in achalasia is poorly understood. This study aims to evaluate morphological changes in esophageal body movement after POEM in type III achalasia by analyzing intraluminal ultrasound (US) images in comparison to type I and II achalasia. METHODS: Intraluminal US images and impedance values of the distal esophagus from 47 achalasia patients who underwent POEM or pneumatic dilatation (PD) (30 patients in the POEM group and 17 patients in the PD group) with pre- and post-procedural high-resolution impedance manometry and intraluminal US examinations were analyzed. The muscle thickness (MT), muscle cross-sectional area, lumen cross-sectional area (LCSA), contractility and distensibility indices, swallow-to-distension interval, and distension duration during each bolus transport were analyzed. RESULTS: The MT increased and LCSA decreased significantly (P < 0.001), but the contractility index was not improved after POEM or PD in type I achalasia. Baseline MT increased and LCSA decreased significantly after POEM and PD in type II achalasia (P < 0.001). In contrast, MT and the swallow-to-distension interval decreased and the distension LCSA/duration and contractility index increased after POEM in type III achalasia (P < 0.001). In contrast to type I and II achalasia, in type III achalasia, these effects were unique to the POEM group. CONCLUSIONS: POEM decreased the esophageal LCSA by decreasing intrabolus pressure without improving contractility in type I and II achalasia. In contrast, POEM increased esophageal body distension and contractility and improved the inhibitory process during bolus transport in type III achalasia.

Olaparib-induced Apoptosis Through EBNA1-ATR-p38 MAPK Signaling Pathway in Epstein-Barr Virus-positive Gastric Cancer Cells.

BACKGROUND: Epstein-Barr virus (EBV)-associated gastric cancer has been identified as a cancer subtype with definitive clinical and molecular characteristics. Although olaparib, a poly ADP ribose polymerase (PARP) inhibitor, is considered a potential effective agent for gastric cancer, the effect and underlying mechanism of olaparib on gastric cancer depending on EBV infection is not fully understood. MATERIALS AND METHODS: EBV-positive SNU719 and EBV-negative SNU638 gastric cancer cell lines were used to identify the effects of olaparib using the trypan blue exclusion method and annexin V staining assay. To observe the underlying cellular signaling mechanisms of olaparib-induced cell death, Epstein-Barr virus nuclear antigen 1 (EBNA1) and signaling related molecule expression were assessed using transfection, silencing of specific genes using small interfering RNA (siRNA), western blotting and signaling inhibition assay. RESULTS: Olaparib decreased the cell viability of EBV-positive SNU719 gastric cancer cells through caspase-3-dependent apoptosis in a dose dependent manner, whereas EBV-negative SNU638 gastric cancer cells showed drug resistance to olaparib. EBNA1 was expressed in SUN719 gastric cancer cells; however, ataxia telangiectasia and Rad3 related (ATR) and phosphorylated ATR kinase were expressed in SNU638 gastric cancer cells. EBNA1 transfection decreased ATR phosphorylation through p38 mitogen-activated protein kinase (MAPK) phosphorylation in SUN638 gastric cancer cells, and silencing of ATR kinase increased the susceptibility of these cells to olaparib treatment. Moreover, VE-821, an ATR kinase specific inhibitor, also increased the sensitivity of SNU638 cells to olaparib. In contrast, SB203580, a p38 MAPK inhibitor, inhibited this increase in sensitivity to olaparib by EBNA1 transfection. CONCLUSION: Olaparib treatment led to different cellular responses depending on EBV infection in gastric cancer cell lines. These results provide new insights into the mechanism of olaparib-induced apoptosis in gastric cancer cells and suggest that EBV infection should be considered when developing new potential therapeutic agents for gastric cancer.

Effects of Epstein-Barr Virus Infection on the Response of Human Breast Cancer Cells to Nicotine.

BACKGROUND/AIM: The purpose of our study was to test whether EBV infection affects the response of human breast cancer cells to nicotine. In addition, the underlying signaling mechanisms were evaluated. MATERIALS AND METHODS: EBV-infected MDA-MB-231 and LMP1-transfected MDA-MB-231 breast cancer cells were established. Reverse transcription-polymerase chain reaction and western blotting were performed to evaluate nicotine receptor expression. To verify the functional role and underlying signaling mechanism of nicotine receptor expression induced by EBV infection, morphologic analysis, and proliferation and inhibition assays were performed. RESULTS: Both EBV infection and LMP1 transfection increased cell proliferation and induced the up-regulation of α9-nAChR expression. Additionally, nicotine treatment induced tumorigenic activity in both EBV-infected and LMP1-transfected MDA-MB-231 breast cancer cells. Western blot and inhibitor assays showed that the nicotine-induced signaling was mediated through MAPK/ERK and AKT signaling pathways in EBV-infected and LMP1-transfected breast cancer cells, respectively. CONCLUSION: These results suggest that EBV infection and EBV-related LMP1 may act as potential molecular targets for breast cancer risk associated with nicotine, and provide a novel insight into the mechanism of nicotine stimulation in EBV-positive breast cancer cells.

Erlotinib Activates Different Cell Death Pathways in EGFR-mutant Lung Cancer Cells Grown in 3D Versus 2D Culture Systems.

BACKGROUND/AIM: Non-small cell lung cancer patients with epidermal growth factor receptor (EGFR) mutation have been shown to have a good response to erlotinib, a receptor tyrosine kinase inhibitor of EGFR. In this study, we found that the cell death pathways activated by erlotinib in 2D and 3D culture systems are different. MATERIALS AND METHODS: The cell death pathways induced by erlotinib were evaluated by flow cytometry and immunoblotting in both 2D and 3D culture systems of EGFR mutant lung cancer cells. RESULTS: Treatment with erlotinib induced caspase 8 activation and up-regulation of TNF-related apoptosis-inducing ligand (TRAIL) expression only in 3D cultures. Knockdown of TRAIL attenuated both erlotinib-induced activation of caspase-8 and apoptosis in 3D cultures. Erlotinib also increased LC3, an autophagy marker, expression and c-Jun N terminal kinase (JNK) activation. Both 3-MA as an autophagy inhibitor and SP600125 as a JNK inhibitor, significantly inhibited erlotinib-induced cell death. CONCLUSION: Erlotinib induces apoptotic cell death in 3D cultures through an autophagy-TRAIL-JNK pathway.

VSIG4 Induces Epithelial-Mesenchymal Transition of Renal Tubular Cells under High-Glucose Conditions.

High glucose-mediated tubular injury contributes to the development and progression of diabetic nephropathy through renal tubulointerstitial fibrosis. V-set immunoglobulin-domain-containing 4 (VSIG4), a B7 family-related protein, is a complement receptor. Although the role of epithelial-mesenchymal transition (EMT) has been reported in several diseases, little is known about its relationship with VSIG4 under diabetic conditions. This study aimed to investigate the role of VSIG4 in human tubule cells stimulated by high glucose (HG, 55 mM). HG upregulated both mRNA and protein levels of VSIG4 in proximal tubule cells (HK-2 cells) and Madin Darby Canine Kidney cells. These upregulations were accompanied by increased expression of mesenchymal markers such as fibronectin, N-cadherin, matrix metalloproteinase 9, and vimentin, and by decreased expression of the epithelial marker, E-cadherin. The siRNA-mediated inhibition of VSIG4 in HK-2 cells restored the dysregulation of EMT in cells. Interestingly, VSIG4 inhibition did not affect the expression of transforming growth factor (TGF)-ß, whereas inhibition of TGF-ß reduced VSIG4 expression, subsequently suppressing fibrosis markers. These findings suggest that VSIG4 plays an important role in mediating renal tubular EMT through the downstream action of HG-induced TGF-ß activation.

Nicotinamide adenine dinucleotide phosphate oxidase inhibitor induces apoptosis on Epstein-Barr virus positive B lymphoma cells.

Over-expression of nicotinamide adenine dinucleotide phosphate oxidase (Nox) isoform enzymes was recently reported in various cancers including Burkitt's lymphoma (BL). However, the functions of Nox isoform enzymes in BL remain poorly understood. In this study, Nox isoform expression and the effects of a Nox-specific inhibitor were evaluated in Epstein-Barr virus (EBV)-positive Raji BL cells in comparison with EBV-negative Ramos BL cells. To evaluate Nox enzyme expression in Raji and Ramos BL cells, polymerase chain reaction (PCR) and western blot analysis were performed. To verify the intracellular signaling mechanism of the Nox inhibitor-induced apoptosis of Raji cells, WST-1 assay, trypan blue exclusion method, flow cytometry, PCR, western blotting, and bromodeoxyuridine staining were conducted. Experiments using the pan-caspase inhibitor z-VAD, reactive oxygen species scavenger N-acetyl-L-cysteine (NAC), and Bim inhibitor 1 were performed. PCR and western blot results showed that Nox isoform enzymes were highly expressed in EBV-positive BL Raji cells compared with EBV-negative BL Ramos cells. The Nox2 inhibitor induced apoptosis of Raji cells in time- and dose-dependent manners. The Nox2 inhibitor also caused up-regulation of Bim and Noxa, down-regulation of Mcl-1, translocation of Bax, release of cytochrome c, and caspase cascade activation, resulting in apoptosis. Furthermore, z-VAD, NAC, and BI-1 effectively blocked the Nox2 inhibitor-induced apoptosis of Raji cells. Taken together, these results provide a novel insight into the mechanism of Nox inhibitor-induced apoptosis and evidence for Nox as a therapeutic target to treat EBV-positive malignancies.

Celecoxib upregulates ULBP-1 expression in lung cancer cells via the JNK/PI3K signaling pathway and increases susceptibility to natural killer cell cytotoxicity.

Lung cancer has the highest cancer mortality rate in the world, and effective therapies are still required. Cyclooxygenase-2 (COX-2) is highly expressed in numerous types of cancer, and is therefore considered a possible target of cancer treatment. Celecoxib, a selective COX-2 inhibitor, has binding pockets that interact with COX-2 and disrupt its enzymatic activities. In addition, celecoxib is able to affect cellular functions in a COX-2-independent manner. The present study aimed to investigate if celecoxib affected natural killer (NK) cell receptors and susceptibility to NK cell toxicity. For this purpose, PCR, immunoblotting, flow cytometry analysis and NK cell cytotoxicity assays were performed. The present study revealed that sublethal concentrations of celecoxib increased the expression levels of UL16-binding protein 1 (ULBP-1), a natural-killer group 2 member D (NKG2D) ligand, in lung cancer A549 and H460 cell lines. ULBP-1 mRNA and protein expression was induced in a dose- and time-dependent manner after celecoxib treatment. Expression levels of other NKG2D ligands, such as ULBP-2, ULBP-3, MHC class I-related chain A (MICA) and MICB did not change considerably compared to ULBP-1 in response to celecoxib treatment. Fluorescence microscopic images revealed abundant ULBP-1 in the cytoplasm after celecoxib treatment. Both JNK and PI3K may be involved in the induction of ULBP-1 expression after celecoxib treatment in A549 and H460 cells. In a NK cytotoxicity assay, celecoxib increased the sensitivity to NK cell-mediated cytotoxicity via interaction with ULBP-1 in lung cancer cells. Overall, the present results demonstrated that celecoxib treatment induced ULBP-1 expression in lung cancer cells, thereby increasing their susceptibility to NK cell cytotoxicity. These results suggest that the effects of conventional anticancer therapy may potentially be enhanced by using celecoxib, which targets COX-2, to enhance the sensitivity of lung cancer cells to NK cell-mediated cytotoxicity.

APX-115A, a pan-NADPH Oxidase Inhibitor, Induces Caspase-dependent Cell Death by Suppressing NOX4-ROS Signaling in EBV-infected Retinal Epithelial Cells.

PURPOSE: Epstein-Barr virus is a γ-herpes virus that infects primary B cells and can transform infected cells into immortalized lymphoblastoid cell lines (LCL). The role of EBV in malignancies such as Burkitt's lymphoma and nasopharyngeal carcinoma is well understood, however, its role in EBV-infected retinal cells remains poorly understood. Therefore, we investigated the effect of EBV on the growth of retinal cells. METHODS: Previously, we established and reported a cell line model to address the relationship between EBV infection and retinal cell proliferation that used adult retinal pigment epithelium (ARPE-19) and EBV infection. To determine the effect of EBV on ARPE-19 cells, cell death was measured by propidium iodine/annexin V staining and reactive oxygen species (ROS) were measured by FACS, and protein expression was evaluated using western blot analysis. Also, downregulation of LMP1 and NADPH oxidase 4 (NOX4) expression was accomplished using siRNA technology. RESULTS: We found that ROS were dramatically increased in EBV-infected ARPE19 cells (APRE19/EBV) relative to the parental cell line. Additionally, the expression level of NOX4, a main source of ROS, was upregulated by EBV infection. Interestingly, downregulation of LMP1, one of the EBV viral onco-proteins, completely decreased EBV-induced ROS accumulation and the upregulation of NOX4. Treatment with APX-115A, a pan-NOX inhibitor, induced apoptotic cell death of only the EBV-infected ARPE19 cells but not the parental cell line. Pretreatment with z-VAD, a pan-caspase inhibitor, inhibited NOX inhibitor-induced cell death in ARPE19/EBV cells. Furthermore, APX-115A-induced cell death mediated the activation of JNK and ERK. Finally, we confirmed the expression level of NOX4, and APX-115A induced cell death of EBV-infected human primary retina epithelial cells and the activation of JNK and ERK. CONCLUSION: Taken together, these our results suggest that APX-115A could be a therapeutic agent for treating EBV-infected retinal cells or diseases by inhibiting LMP1-NOX4-ROS signaling.

Ampelopsin Induces DR5-Mediated Apoptotic Cell Death in EBV-Infected Cells through the p38 Pathway.

Ampelopsin (AMP) is a well-known flavonoid that exerts a number of biological and pharmacological effects including anticancer effects against several cancer cell lines. In this study, we investigated the anticancer activity of AMP against Epstein-Barr virus (EBV)-positive cells and its mechanism of action. Our results showed that AMP dose-dependently inhibited cell viability and induced apoptotic cell death in EBV-positive cells without cytotoxicity in EBV-negative cells. In particular, AMP induced caspase-8 dependent apoptosis via upregulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and death receptor (DR5). Knockdown of DR5 by RNA interference blocked AMP-induced apoptosis. Furthermore, AMP dose-dependently activated p38 mitogen-activated protein kinases (MAPKs) in EBV-positive cells. Additionally, SB203580 (a p38-MAPK inhibitor) effectively inhibited apoptotic cell death. These results demonstrate that treatment with AMP induces the apoptosis of EBV-positive cells through upregulation of TRAIL/DR5 and activation of p38 signaling. Therefore, these results provide experimental information for developing AMP as a new therapeutic drug against EBV-positive cancer.